1. Field of the Invention
The present invention relates to a method of the production of a nanoparticle in which a cavity of a protein is utilized, a nanoparticle produced by the method of the production, and a nanoparticle-protein complex generated in the production step of the method of the production.
2. Description of the Related Art
Mainstream of the development of functional materials implemented so far involves exploration and synthesis of novel compounds which allow performing a desired function. However, in recent years, it has been desired to allow performing new functions which can not be achieved in a bulk state through producing nanoparticles obtained by fine division of a substance into a nanometer size. In particular, production of nanoparticles of semiconductors or inorganic materials including a metal compound as a center has been strongly desired for the purpose of producing quantum effect devices which draw the attention in recent years.
Methods of the production of nanoparticles which have been conventionally carried out involve physical grinding methods, chemical synthesis methods and the like. For example, the physical grinding methods have been widely used in order to obtain starting materials upon baking of ceramics. In addition, examples of the chemical synthesis method include methods in which gold nanoparticles are produced through reducing chloroauric acid among long chain organic compounds. The long chain organic compound in this process inhibits the growing of a gold particle to an enormous size.
Furthermore, there exist methods in which a complex between an organic compound and a nanoparticle is generated followed by a chemical reaction to result in uniform particles. As an application of this method, there also exists a method in which gold nanoparticles are obtained having a self-assembled monomolecular membrane (SAM membrane) formed on their surfaces through fixing a gold atom on a material for forming a SAM membrane, and assembling the material such that the gold atom becomes the center. Moreover, a method is also executed in which a micelle including a material which forms a nanoparticle is produced, and nanoparticles are produced using a chemical reaction in the micelle.
In order to produce a quantum effect device, it is essential to obtain nanoparticles having the identical diameter. Energy level which can be attained by an electron in a nanoparticle greatly varies depending upon the diameter in the order of several nanometers. Hence, to give a constant diameter is important in applying nanoparticles having a quantum effect to an electronic circuit.
However, it is difficult to obtain nanoparticles having a uniform diameter by the conventional methods as described above. In the physical grinding method, for example, it is originally difficult to make the particle diameter smaller than the micron size, and even though the size could approximate the order of nanometer, no mechanism has been established to accomplish a constant diameter. Hence, great spread of the diameter of thus resulting nanoparticles is inevitably generated. In addition, in the chemical synthesis method, the great spread of the diameter of the resulting nanoparticles is also generated inevitably because a chemical reaction is utilized therein. Further, it is also disadvantageous in respect of the required time period and cost.
On the other hand, in an attempt to apply biotechnology to other filed, there exist investigations in which nanoparticles having uniform size in the order of nano are intended to be produced through rendering the incorporation of a metal or a metal compound into apoferritin that is a protein having a function to hold a metal compound. Investigations have been carried out so that any of various kinds of metals or metal compounds is introduced into apoferritin in compliance with the use of the nanoparticle.
Apoferritin is now explained below. Apoferritin is a protein which is present widespread in an animate nature, and plays a role to regulate the amount of iron which is an essential trace element in a living body. A complex of iron or an iron compound with apoferritin is referred to as ferritin. Since iron is deleterious to a living body when it is present in an excessive amount in the body, excess iron is stored in the body in the form of ferritin. Furthermore, ferritin returns to apoferritin through releasing an iron ion as needed.
FIG. 1 is a schematic view illustrating the structure of apoferritin. As shown in FIG. 1, apoferritin 1 is a spherical protein having the molecular weight of about 460,000 with 24 monomer subunits, which are formed from a single polypeptide chain, being assembled via noncovalent bonds, having the diameter of about 12 nm, and exhibits higher thermostability and pH stability in comparison with general proteins. There is a cavity-like holding part 4 having the diameter of about 6 nm in the center of apoferritin 1, and the outside and the holding part 4 are connected via a channel 3. For example, when a bivalent iron ion is incorporated into apoferritin 1, the iron ion enters from the channel 3, and reaches to the holding part 4 after being oxidized in a place which is present within a part of the subunits and is referred to as a ferrooxidase center (iron oxidation active center). The iron ion is thereafter concentrated in a negative charge region on the inner surface of the holding part 4. Then, the iron atoms assemble by 3000 to 4000, and held in the holding part 4 in the form of a ferrihalide (5Fe2O3.9H2O) crystal. Diameter of the nanoparticle, which was held in the holding part 4, comprising the metal atom is nearly equal to the diameter of the holding part 4, which is about 6 nm.
Using this apoferritin, nanoparticle-apoferritin complexes have been generated in which a metal or a metal compound other than iron is artificially carried.
Introduction of a metal or a metal compound such as manganese (see, P. Mackle, 1993, J. Amer. Chem. Soc. 115, pp. 8471–8472; and F. C. Meldrum et al., 1995, J. Inorg. Biochem. 58, pp. 59–68), uranium (see, J. F. Hainfeld, 1992, Proc. Natl. Acad. Sci. USA, 89, pp. 11064–11068), beryllium (see, D. J. Price, 1983, J. Biol. Chem. 258, pp. 10873–10880), aluminum (see, J. Fleming, 1987, Proc. Natl. Acad. Sci. USA, 84, pp. 7866–7870), and zinc (D. Price and J. G. Joshi, Proc. Natl. Acad. Sci. USA, 1982, 79, pp. 3116–3119) into apoferritin has been reported so far. The diameter of the nanoparticles including any one of these metals or metal compounds is also nearly equal to the diameter of the holding part 4 of apoferritin, which is about 6 nm.
Summary of the process in which nanoparticles including an iron atom are formed in apoferritin in the natural world is as follows.
On the surface of the channel 3 which connects between the outside and inside of apoferritin 1 (see, FIG. 1) are exposed amino acids having a negative charge under a condition of pH of 7–8, thus iron (II) ions having a positive charge are incorporated into the channel 3 by an electrostatic interaction. On the inner surface of the holding part 4 of apoferritin 1 are exposed a lot of glutamic acid residues which are amino acid residues having a negative charge at pH 7–8, similarly to the inner surface of the channel 3, and the iron (II) ions incorporated from the channel 3 are oxidized at the ferroxidase center, followed by introduction to the internal holding part 4. Then, the iron ions are concentrated by an electrostatic interaction, and the core formation of a ferrihalide (5Fe2O3.9H2O) crystal is caused.
Thereafter, a core which comprises iron oxide is grown through adherence of the iron ions, which are sequentially incorporated, to the crystal core, and accordingly, a nanoparticle having the particle diameter of 6 nm is formed in the holding part 4. Summary of the formation of a nanoparticle including iron oxide as well as the incorporation of the iron ions is as set forth above.
Although a mechanism of the incorporation of iron ions into apoferritin was described hereinabove, it is believed that other metal ions, which were reported hitherto as candidates for the introduction, also involve an approximately similar mechanism to that for the iron ion. However, metal ions which can be incorporated in the cavity part by the method described above were limited to particular ones.